1. Field of the Invention
The present invention is based on the unexpected findings that exosomes released to the extracellular milieu can carry selective RNA from the parental cells. This can, according to this invention, be used to transfer genetic material to recipient cells by exosomes. By transferring nucleic acids to recipient cells, exosomes affect another cell's (recipient cells) protein machinery and thus protein content, and the invention demonstrates for the first time that nucleic acids, for example RNA and DNA, deliberately can be transferred between the cells or organs using exosomes, and can thus be utilized for gene modulation and therapy in mammalian cells.
2. Description of the Related Art
Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges between 30 and 100 nm in diameter. Their surface consists of a lipid bilayer from the donor cell's cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface.
Exosomes exhibit different composition and function depending on the cell type from which they are derived. There are no “exosome-specific” proteins; however several proteins identified in these vesicles are associated with endosomes and lysosomes reflecting their origin. Most exosomes are enriched in MHC I and II (major histocompatibility complex I and II; important for antigen presentation to immunocompetent cells such as T-lymphocytes), tetraspanins, several heat shock proteins, cytoskelatal components such as actins and tubulins, proteins involved in intracellular membrane fusion, signal transduction proteins and cytosolic enzymes.
Exosomes are produced by many cells including epithelial cells, B and T lymphocytes, mast cells (MC) as well as dendritic cells (DC). In humans, exosomes have been found in blood plasma, urine, bronchoalveolar lavage fluid, intestinal epithelial cells and tumor tissues.
All functions of exosomes have not been elucidated, but data strongly indicates they mediate communication between cells. This communication could take place in different ways. First, exosomes could bind to cell surface receptor in a similar way as cell to cell interaction. Second, exosomes could attach to the cell membrane and give the cells new receptors and properties. Thus, exosomes can also fuse with target cells and exchange membrane proteins and cytosol between two cell types.
We have put extra effort into understanding the content and biological function of exosomes specifically released by mast cells. In proteomic assays we have found that these exosomes contain a larger number of proteins than previously understood. However, the unique finding from our research is that we have discovered a substantial amount of selective RNA in exosomes from mast cells. Furthermore, use of different recipient cells displays an uptake of exosomal RNA indicating transfer of genetic material from exosomes into recipient cells, which in turn will lead to translation of specific protein in the target cells.
Considering the exosomal protein content and their capacity to communicate with different recipient cells, it is particularly useful to be able to modify the genetic content of exosomes in order to add or regulate a gene in recipient cells. The method using the exosomes' capacity of carrying specific genetic material and transferring it to recipient cells is described in this application. In this method, the recipient is affected in its function, as well as in its ability to stay alive, further develop, proliferate or mature.
The method is unique and different from any previous described methods. Several patents and patent applications use exosomes to influence the immune system through stimulatory or inhibitory function via exosomal protein interaction with immune cells, and for treatment of viral disease by influencing the immune system. It has been suggested that exosomal proteins can be modified by mutation to affect the immune system. However, no patent or patent application or any publicly available information that we have found describes or suggests use of exosomes to transfer genetic material or nucleic acids to cells.